Fatty acid biosynthesis in higher plants has recently attracted increased interest because of the possible use of plant oils as renewable sources for reduced carbon. The diversity of fatty acid forms in wild plants is vast compared to that of crop plants. This diversity is reflected in the variations in chain length, the number and position of double bonds, and the position and occurrence of a variety of other functional groups in the fatty acids of wild plants.
In plants, fatty acid biosynthesis occurs in the chloroplasts of green tissue or in the plastids of non-photosynthetic tissues. The primary products in most plants are acyl carrier protein (ACP) esters of the saturated palmitic (palmitoyl-ACP) and/or stearic (stearoyl-ACP) acids, palmitic acid having a 16 carbon atom chain length and stearic acid having an 18 carbon atom chain length. Two types of desaturase molecules are involved in the production of monounsaturated fatty acids (monoenes), soluble, and integral membrane proteins. Desaturases are specific for a particular substrate carbon atom chain length (chain length specificity) and introduce the double bond between specific carbon atoms in the chain (double bond positional specificity) by counting from the carboxyl end of the fatty acid. For instance, the castor xcex949-18:0 desaturase is specific for stearoyl-ACP, and introduces a double bond between carbon atoms 9 and 10.
The introduction of non-native desaturase isoforms having unique characteristic chain length and double bond positional specificities into agricultural crops offers a way to manipulate the content, physical properties and commercial uses of plant-produced oils. Unfortunately, the introduction of non-native acyl-ACP desaturase isoforms into agricultural crop plants has yet to lead to the efficient production of unusual monoenes by agricultural crop plants. An alternative way in which to accomplish the manipulation of the content, physical properties and commercial uses of oilseed crops would be through the introduction of a native desaturase which had been manipulated in such a way as to alter its chain length and/or double bond positional specificities.
As the genes encoding more desaturase enzymes are identified it is becoming apparent that many of the different activities are derived from relatively few common archetypes encoding the soluble and membrane classes of desaturases. Molecular modeling and X-ray crystallographic studies of soluble acyl-ACP desaturases have identified amino acid residues within the substrate binding channel which are in very close proximity to the fatty acid substrate. Such residues are referred to as xe2x80x9ccontact residuesxe2x80x9d. That earlier research demonstrated that certain modifications of one or more contact residues and modification of some non-contact residues can alter the chain-length and double bond positional specificities of acyl-ACP desaturases in vitro (Cahoon, et al. Proc. Natl. Acad. Sci. USA (1997) 94:4872-4877 and Cahoon, et al. U.S. Pat. Nos. 5,705,391, 5,888,790 and 6,100,091). Those studies were carried out using predictions formulated from the three dimensional structure of the castor xcex949-18:0 acyl-ACP desaturase in combination with alignment of its sequence with that of a xcex946-16:0 acyl-ACP desaturase as well as with the sequences of other 18:0 desaturases. The studies examined the effects of replacing specific contact and non-contact amino acid residues of one enzyme with the amino acid in the cognate position of the other enzyme on the in vitro substrate chain length and double bond positional specificities of several desaturase enzymes. The studies demonstrated that substituting a major portion of the substrate binding channel of a xcex949-18:0 desaturase into the homologous position of a xcex946-16:0 desaturase converted its in vitro specificity to that of a xcex949-18:0 desaturase. This could also be accomplished by replacing five specific amino acids of the xcex946-16:0 desaturase with five amino acids of the xcex949-18:0 desaturase which occupy homologous positions. It was also shown that substituting bulky amino acids (isoleucine for proline at position 179 and phenylalanine for leucine at position 118) into the substrate binding channel of the xcex949-18:0 desaturase increased its preference for the 16:0-ACP substrate such that the in vitro 16:0-ACP activity became slightly more than two-fold greater than its remaining 18:0-ACP activity.
The ability to manipulate the chain length and double bond position specificities of desaturases has great potential with regard to generation and use of mutated native desaturases in the production of commercially useful products, such as vegetable oils rich in monounsaturated fatty acids. Such vegetable oils are important in human nutrition. In addition, because a double bond in an otherwise saturated carbon chain is readily susceptible to chemical modification, fatty acid chains having double bonds in unique positions produced by crop plants can be useful raw materials for industrial processes.
The earlier studies making use of molecular modeling and crystallographic data, while successful, were extremely time consuming and the in vitro activity of the altered enzymes was not directly correlated to the in vivo specificities of the altered enzymes. Those studies pointed out a need for a simplified and general method for readily producing mutants of desaturases which have altered and desirable chain length and double bond positional specificities.
The present invention relates to a simple and general method for producing a mutant of a fatty acid desaturase, the original desaturase having an 18 carbon atom chain length substrate specificity, the mutant produced having substantially increased activity relative to the original desaturase towards fatty acid substrates with chains containing fewer than 18 carbons. The method involves inducing one or more mutations in the nucleic acid sequence encoding the original desaturase, transforming the mutated nucleic acid sequence under conditions for expression into a cell which normally requires a growth medium that is supplemented with unsaturated fatty acids in order to proliferate (i.e., an unsaturated fatty acid auxotroph cell), and then selecting for recipient cells which have received a mutant fatty acid desaturase with a specificity for shorter carbon atom chain length substrates. In a preferred embodiment, the mutated nucleic acid sequences are transformed into an E. coli unsaturated fatty acid auxotroph designated MH13. The cells are then grown in the absence of added unsaturated fatty acids to select for recipient MH13 cells which express mutated enzymes which are capable of producing sufficient unsaturated fatty acids in the cell to support growth, thereby overcoming the auxotrophy. Other aspects of the present invention include the mutants which are produced. Mutants of castor xcex949-18:0-ACP desaturase produced by the method arise from amino acid substitutions at specific residues. These mutants each have altered substrate chain length specificity, of 16- or fewer carbon atoms. Other embodiments of the present invention encompass the expression of the mutant desaturase molecules in individual cells and also in transgenic plants, for the production of specific fatty acid products.